摘要
目的:建立同时测定人血浆中氯吡格雷(CLP)及其中间代谢产物2-氧-氯吡格雷(2-O-CLP)、非活性代谢产物氯吡格雷羧酸代谢物(CLPCA)和活性代谢产物氯吡格雷硫醇代谢物(CLPTM)浓度的方法。方法:选取陆军军医大学第一附属医院确诊为脑卒中的90名患者,早晨空腹口服1片氯吡格雷片(75 mg/片),于服药2 h后采集血样,将CLPTM经2-溴-3’-甲氧基苯乙酮衍生形成CLPTM-D后与其余3种待测物一起通过乙腈蛋白沉淀提取后,采用液相色谱-串联质谱法(LC-MS/MS)测定其浓度。色谱柱为Agilent poroshell 120 EC-C18,流动相为0.1%甲酸的乙腈溶液-0.1%甲酸水溶液(90∶10,V/V),采用多反应监测模式进行正离子检测,检测离子对分别为CLPCA质荷比(m/z)308.1→198.1、CLP m/z 322.3→212.0、2-O-CLP m/z 338.3→155.0、CLPTM-D m/z504.4→354.1、内标噻氯吡啶m/z 264.0→154.1。结果:CLPCA、CLP、2-O-CLP、CLPTM-D和内标的保留时间分别为2.01、3.32、2.83、2.68、1.87 min,CLPCA、CLP、2-O-CLP、CLPTM-D检测质量浓度的线性范围分别为100~10000、0.2~20、0.3~30、0.5~50ng/mL(r均≥0.9995),日内、日间精密度试验的RSD均≤9.5%(n=5),准确度为93.5%~98.9%(n=5),提取回收率为85.4%~95.9%(n=5),基质效应的CV为2.7%~6.2%(n=5)。稳定性(-80℃放置3个月、3次冷冻-解冻循环、4℃放置8 h)试验中,CLPCA、CLP、2-O-CLP和CLPTM-D的RE均≤10.0%(n=5)。结论:建立的LC-MS/MS法特异性强,测定结果准确可靠,可用于检测人血浆中CLP及其3种代谢产物的浓度。
OBJECTIVE:To establish the method for simultaneous determination of clopidogrel(CLP),its intermediate metabolite(2-O-CLP),inactive metabolite(CLPCA)and active metabolite(CLPTM)in human plasma.METHODS:Totally 90 patients diagnosed as stroke were selected from the First Affiliated Hospital of Army Medical University.They were given one CLP tablet(75 mg/tablet)orally on an empty stomach in the morning.Blood samples were collected 2 h after taking the tablet.CLPTMD was formed by derivation of CLPTM with 2-bromo-3’-methoxyacetophenone and extracted by precipitation of acetonitrile protein together with the other three substances to be measured.LC-MS/MS method was adopted.The determination was performed on Agilent poroshell 120 EC-C18 column with mobile phase consisted of acetonitrile(0.1%formic acid)and water(0.1%formic acid)(90∶10,V/V).The quantitation analysis was performed using multiple reaction monitoring at the specific ion transitions of m/z308.1→198.1(CLPCA),322.3→212.0(CLP),338.3→155.0(2-O-CLP),504.4→354.1(CLPTM-D)and 264.0→154.1(ticlopidine,internal standard),respectively.RESULTS:The retention time of CLPCA,CLP,2-O-CLP,CLPTM-D and internal standard were 2.01,3.32,2.83,2.68,1.87 min,respectively.The linear range of CLPCA,CLP,2-O-CLP and CLPTM-D were100-10000,0.2-20,0.3-30,0.5-50 ng/mL(all r≥0.9995).The intra-day and inter-day RSD were all less than 9.5%(n=5).Accuracy ranged from 93.5%-98.9%(n=5),and extraction recovery was from 85.4%to 95.9%(n=5).The matrix effect ranged from 2.7%-6.2%(n=5).In stability tests(storing at-80℃for 3 months,3 freeze-thaw cycles,storing at 4℃for 8 h),RE of CLP,CLPCA and CLPTM-D were all lower than 10.0%(n=5).CONCLUSIONS:Established LC-MS/MS method has the advantages of high specificity,accuracy and reliability,and can be used to detect the concentration of CLP and its three metabolites in human plasma.
作者
刘职瑞
姚璞
杨波
王瑜
冯伟
孙凤军
LIU Zhirui;YAO Pu;YANG Bo;WANG Yu;FENG Wei;SUN Fengjun(Dept.of Pharmacy,the First Affiliated Hospital of Army Medical University,Chongqing 400038,China)
出处
《中国药房》
CAS
北大核心
2019年第21期2946-2951,共6页
China Pharmacy
基金
重庆市社会事业与民生保障科技创新专项(No.cstc2016shms-ztzx10004)